The objective of this investigation was to determine the role of Pyk2, an intracellular nonreceptor protein tyrosine kinase for postadhesive inflammatory cell migration, on airway inflammation and hyperresponsiveness in immune-sensitized mice. Blockade of Pyk2 was effected by intraperitoneal administration of dominant-negative C-terminal Pyk2 fused to a TAT protein transduction domain (TAT-Pyk2-CT). Ovalbumin challenge elicited infiltration of both eosinophils and lymphocytes into airways, increased mucus-containing epithelial cells, and caused increased airway hyperresponsiveness to methacholine in immune-sensitized mice. Pretreatment with 10 mg/kg TAT-Pyk2-CT intraperitoneally blocked all of these effects and further decreased secretion of Th2 cytokine IL-4, IL-5, and IL-13 into the bronchoalveolar lavage fluid. Intranasal administration of IL-5 caused eosinophil migration into the airway lumen, which was attenuated by systemic pretreatment with TAT-Pyk2-CT. In each paradigm, treatment with control protein TAT-GFP had no blocking effect. We conclude that Pyk2, which is essential for inflammatory cell migration in vitro, regulates airway inflammation, Th2 cytokine secretion, and airway hyperresponsiveness in the ovalbumin-sensitized mice during antigen challenge in vivo.